Mycobacterium tuberculosis (Mtb) is an extremely successful pathogen that remains a key public health problem, infecting one-third of the human population and causing ~1.5 million deaths a year. The macrophage serves as a major host cell for Mtb, and the bacterium's ability to turn the phagosome into a replicative niche is critical for the establishment and maintenance of chronic infection. This is dependent on Mtb sensing and responding appropriately to intraphagosomal signals, and the research proposed here builds on my discovery of chloride (Cl-) as a novel environmental cue that Mtb responds to during infection. I have further identified a Cl- channel in Mtb, and observe that a Mtb mutant deficient in this channel is severely attenuated for colonization in a murine infection model. My studies raise the novel concept that the sensing and/or maintenance of an appropriate balance of an abundant ion like Cl- can profoundly impact a pathogen's ability to establish a successful infection, and suggest that this aspect of Mtb physiology represents a new target for the development of original and potentially potent tuberculosis treatment strategies synergistic with current approaches. Here, I seek to (i) elucidate the regulatory network governing Mtb's sensing and response to Cl-, using both genetic and chemical biology approaches that exploit my Cl--responsive reporter Mtb strain, and (ii) examine how environmental cues, like Cl-, can impact on Mtb's replication status, using genome-wide mutational analyses together with a novel replication reporter Mtb strain that I recently constructed. I have a longstanding dedication to research, and my career goal is to direct my own research group at an academic institution. My research will take a multi-disciplinary approach to probe host-pathogen interactions especially from the pathogen's perspective, to understand fundamental aspects of how it can colonize its specific microniche, and exploit this knowledge to examine how these critical nodes may be manipulated to shift the balance during infection in favor of the host. The mentored phase of this award will allow me to expand my methodological repertoire, and further develop my research skills and research program. Completion of genetic and chemical screens focused on elucidating Mtb's Cl- regulatory network will further provide me with tools (Mtb mutants and chemical probes that modulate Mtb's Cl- response) and results from which new projects will be developed as I transition to an independent position. During the independent phase, I will also begin to address Mtb's sensing and response to specific environmental cues in a different conceptual framework, in particular interrogating how it may be linked to regulation of bacterial replication, independent of nutrient availability. The mentored phase of this award will be carried out in the lab of Dr. David Russell at Cornell University. The Russell lab and Cornell University provide an ideal environment for my research and training, with unique resources that will enable me to carry out the proposed research, and continue to acquire skills in other facets crucial to my goal of becoming a successful independent investigator, such as lab management, teaching, and scientific communication skills. In addition, I have assembled an advisory committee with diverse expertise in Cl- channels and transporters, in chemical biology underlying signal transduction systems, and in Mtb replication and single cell imaging, to provide further support during the K99 phase and as I transition to the R00 phase. The research proposed here integrates genetic and chemical biology studies that will shed light on the interplay between host cellular environmental cues and Mtb's establishment and maintenance of infection, insights essential to understanding Mtb's life cycle, and for identifying potential targts for the development of new synergistic treatment methods. Combined with other aspects of the research and career development plan proposed, it will ensure that I have both the skill set and groundwork data needed to successfully accomplish my goals of establishing my own lab and pursuing microbial pathogenesis research using a multi-disciplinary approach.